Although a number of genomic and biochemical technologies are now used to elucidate the mechanisms of action of bioactive small molecules, affinity-based isolation of molecular targets is a classic, but still powerful, approach. This review highlights recent cases where biochemical isolation of target proteins of bioactive small molecules highlighted general strategies for a successful isolation and identification of molecular targets. This review is intended to be both an update on the most recent findings for those already active in the field of forward chemical genetics and a guide for scientists entering this burgeoning field.